The invention discloses a hydrogel microsphere-filled hybrid membrane capable of adjusting membrane water content as well as preparation and application thereof. The hybrid membrane consists of hydrogel microspheres and polyimide resin. The preparation process of the hybrid membrane comprises the following steps: preparing the hydrogel microspheres; preparing a polyimide resin solution; feeding the hydrogel microspheres into the polyimide resin solution to prepare a casting membrane solution, and preparing the hybrid membrane. The hybrid membrane has the advantages that the preparation process is simple, and the membrane water content can be simply and conveniently adjusted; the prepared hydrogel microsphere-filled hybrid membrane is even in distribution in the membrane; the compatibility between filled particles and a polymer matrix is better; the water absorption of the hybrid membrane is improved and adjusted by the hydrogel microspheres in the hybrid membrane; the prepared hybrid membrane can be used for separating CO2/CH4, and is better in separating property under the wet testing condition; the CO2 permeability coefficient is 270 barrer, and the CO2/CH4 separation selectivity is 59.